Project summary Our goal is to understand novel functions of the platelet glycoprotein (GP) Ib-IX-V complex, a key receptor for several ligands mediating, among others, adhesive interactions (mostly through von Willebrand factor), cell-cell contacts (interacting with leukocyte integrin ?M?2 and P-selectin) and coagulation (binding several coagulation factors including thrombin). With this project we intend to extend the understanding of GPIb physiopathologic relevance beyond megakaryocyte/platelet domains and more firmly into the area of innate immune mecha- nisms. Nonetheless, von Willebrand factor (VWF) and thrombin, key GPIb partners in hemostasis and throm- bosis, remain the main ligands of interest for the proposed new studies. In our work over nearly 30 years we have contributed to establish the bases for understanding how VWF, thrombin and GPIb?, the main subunit in the GPIbIX-V complex, interact. With respect to thrombin, even with the structure of the GPIb?-thrombin com- plex unveiled at atomic level detail, elucidating dependent functions has remained elusive. With the knowledge acquired through past studies and the ensuing generation of mice lacking thrombin-GPIb binding on platelets, and stimulated by surprising preliminary results obtained using these models, we propose two articulated spe- cific aims to develop a project addressing unexpected new functions. In aim 1 we will extend the effort to define the physiopathological significance of thrombin binding to platelet GPIb? developing two sub-aims. In the first, we will explore the functional roles of the newly identified high and low affinity thrombin-GPIb? binding modes, using for the purpose a mouse strain expressing a mutant human GPIb? (Y279F) in which high affinity throm- bin binding is lost but low affinity is retained. In the second, we will address the problem posed by the se- quence divergence between mouse and human GPIb? in the region of thrombin binding. We have identified the residues of mouse GPIb? required for thrombin binding and propose to generate a mouse model with en- dogenous mouse GPIb? selectively defective in thrombin binding but retaining all other ligand interactions with intact species specificity. This will be an essential tool to ascertain the value for human physiopathology of studying mechanisms of thrombin-induced platelet activation in mouse models with different PAR expression on platelets. In aim 2 we will characterize expression and function of GPIb? on mast cells and its functional role in anaphylaxis. We will develop two sub-aims. In the first we will characterize the structure of mast cell ex- pressed GPIb in relation to the other components of the GPIb-IX-V complex. In the second we will explore functions of mast cell expressed GPIb? evaluating roles in cell adhesion and in response to thrombin stimula- tion. We have already generated several new mouse models with defective GPIb expression on mast cells. The results of these studies will advance the understanding of mechanisms linking the function of platelets in hemostasis to relevant processes in inflammation, infection and innate immunity.